Methods to prevent and treat diseases

ABSTRACT

This invention discloses methods and compositions that can treat a variety of tissue injuries and infections. Tissue-derived leukocyte chemotactic factors are rapidly released after injury to mammalian tissue and can act as the initial signal leading to the initiation and amplification of acute and chronic inflammation associated with injury and infection. The present invention generally provides methods and compositions to prevent and treat injury of cells, tissue, or organs by blocking or inhibiting the release of leukocyte chemotactic factors, by administering certain effective compositions to the tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application relies on the disclosures of and claims the benefit ofthe filing dates of U.S. provisional patent application No. 60/818,806,filed 5 Jul. 2006, U.S. provisional patent application No. 60/835,748,filed 3 Aug. 2006, U.S. provisional patent application No. 60/846,684,filed 21 Sep. 2006, and U.S. provisional patent application No.60/876,457, filed 20 Dec. 2007, the entire disclosures of all of whichare hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of human and veterinarianmedicine. More specifically, the invention relates to prevention andtreatment of tissue and organ injury and infection in animals, such asmammals.

2. Discussion of Related Art

Tissue-derived leukocyte chemotactic factors (LCFs) are a group ofapproximately 3 KDa peptides that are rapidly released by local tissues(within about 5 minutes) in response to injury induced by, for example,chemical agents, such as hydrogen peroxide, sodium hydroxide, citricacid, and alcohol. They are also release in response to physical trauma,such as: scraping; vitamin A deficiency; ultraviolet exposure; ischemia;shear stress; viral infection; and endotoxin treatments. Tissue-derivedLCFs were isolated from injured tissues both in vitro using organculture and cell culture, as well in vivo using animal models andpatients with various diseases. Unique tissue-specific LCFs are known tobe released by corneal, conjunctival, retinal, heart, coronary arteries,vessels, urinary bladder, brain, spinal cord, and gastric tissues inresponse to injury.

LCFs are inflammatory mediators that (a) recruit leukocytes from thecirculation into sites of infection or tissue injury, (b) stimulate thesecretion of adhesion molecules by leukocytes and vascular endothelialcells and accordingly increase the adhesion of cells to the site ofinjury, and (c) activate leukocytes and vascular endothelial cells torelease chemokines, cytokines, and toxic agents such as oxygenmetabolites and digestive enzymes.

Various groups have studied LCFs and the inflammation process. Forexample, U.S. Pat. No. 5,403,914 (herein incorporated by reference)discloses the release of a leukocyte chemotactic factor (LCF) fromcardiac tissue in response to injury. The LCF represents the initialsignal that recruits leukocytes to the injured tissue. Furthermore, U.S.Pat. No. 5,091,404 (herein incorporated by reference) discloses a systemusing cyclocreatine to preserve and/or restore the physiologicalfunctionality of myocardial tissue subject to ischemia, and particularlytissue subject to reperfusion. The system imparts to the cardiac tissuethe ability to sustain high levels of adenosine triphosphate or at leastdelay the depletion of adenosine triphosphate (ATP) during totalischemia. It delays the development of acidosis and enhances the promptrecovery of tissue function, such as contractility, in such muscletissue during and following post-ischemic reperfusion.

An inflammatory reaction within tissue is generally characterized byleukocyte infiltration, edema, redness, pain, neovascularization (inadvanced cases), and finally impairment of function. Neutrophils are themajor inflammatory cells in acute inflammation, while mononuclear cellsare the major cells in chronic inflammation. Acute and chronicinflammation are documented to occur after diverse types of tissueinjury. When inflammation is controlled, it provides a central hostdefense. Uncontrolled inflammation, on the other hand, can causepotentially destructive biological responses.

Leukocyte-mediated cell injury is believed to be a major mechanism oftissue injury in acute and chronic inflammation. Leukocytes releasecytotoxic compounds, such as reactive oxygen metabolites (e.g., hydrogenperoxide, superoxide anion, and hydroxyl radicals) and digestiveproteolytic enzymes (e.g., collagenase and elastase). In the case ofreperfusion after ischemia, neutrophils could also be deleterious toinjured tissues because of their large size. Neutrophils can plug tissuecapillaries during reperfusion resulting in what is known as the“no-reflow” phenomenon with resultant impaired perfusion. Furthermore,activated neutrophils induce extended cell injury during “earlyreperfusion” after ischemia.

There is a need in the art to provide compositions and methods toinhibit both tissue inflammation and tissue apoptosis. For treatment andprevention of injuries to organs, there is a need to providecompositions and methods to inhibit cytokine storms and organ apoptosis.There is furthermore a need to improve treatment of tissue infections.For example, a current treatment for anthrax infection involves the useof several different antibiotics, used in combination with vaccines. Newtherapeutic approaches are necessary to better protect anthrax-infectedpatients from vasculitis, vascular and tissue apoptosis, edema, as wellas tissue damage. There is a need in the art to improve treatment ofinfections in general.

Vaccines and viral medications are the two most common approachesgenerally used to prevent and treat viral infections, but neither cancontrol the excessive host inflammatory response including cytokinestorms, which occur secondary to Avian viral influenza infections andcan cause death.

What is particularly needed are methods and compositions that can treata wide variety of tissue injuries across different mammalian tissues aswell as different types, severities, and durations of tissue injuries.

SUMMARY OF THE INVENTION

Tissue-derived leukocyte chemotactic factors are rapidly released afterinjury to mammalian tissue and can act as the initial signal leading tothe initiation and amplification of acute and chronic inflammationassociated with injury and infection. The present invention generallyprovides methods and compositions to prevent and treat tissue injury byblocking or inhibiting the release of leukocyte chemotactic factors. Inthis invention, by “injury” of mammalian cells, tissue, or organs ismeant all possible sources of damage or harm to the structure orfunction of such cells, tissue, or organs.

In a first aspect, the invention provides a method of treating animaltissue, such as mammalian tissue, that has been subject to injury. Ingeneral, the method can comprise: administering to the animal at leastone tissue-protective agent in an amount that is effective for impartingto a tissue an anti-inflammatory response, an anti-apoptotic response,or both anti-inflammatory and anti-apoptotic responses.

In another aspect, the invention provides a tissue-protective agent. Thetissue-protective agent can comprise one or more agents having thedesired activity. For example, the agent can be spinorphin, tynorphin,leuhistin, nimbidin, t-Boc-Phe-D-Leu-Phe-D-Leu-Phe,t-Boc-Methionyl-Leucyl-Phenylalanine, Carbobenzoxy-Phe-Met, Substance Pantagonist R(dextro-)PKP (dextro-)FQ(dextro-)WF(dextro-)WLL-NH₂,Cyclosporine H, or pentoxifylline. The tissue-protective agent can alsoinclude one or more of a variety of anti-oxidants, such as ascorbic acidor lycopene.

Among other things, the tissue-protective agent can comprise an antibodyagainst formyl peptide receptors that can bind to formylated ligands, oran antibody against formyl peptide receptors that can bind tonon-formylated ligands. The tissue-protective agent of the invention cancomprise a plurality of soluble formyl peptide receptors that can bindto formylated ligands, or a plurality of soluble formyl peptidereceptors that can bind to non-formylated ligands. The agent can alsocomprise an antibody against a tissue-derived leukocyte chemotacticfactor.

The tissue-protective agent can comprise a creatine analogue, such ascyclocreatine, a salt of cyclocreatine (e.g., cyclocreatine phosphate),or other known creatine analogues or molecular entities with the same orsimilar function to creatine analogues.

In some embodiments relating to creatine analogues, a method of treatinganimal (e.g., mammalian) tissue subject to injury comprises the step ofadministering to the mammal a creatine analogue in an amount between0.01 g and 0.1 g creatine analogue per kg of mammal body weight. Incertain embodiments, the administered dose of creatine analogue is inthe 0.03-0.08 g/kg range. In these embodiments, the creatine analogue(e.g., cyclocreatine and cyclocreatine phosphate) can reduceintracellular cAMP production in the tissue. Tissue apoptosis can besignificantly reduced or eliminated.

The tissue-protective agent can comprise, among other things, ametabolite of the mitochondria of the mammalian tissue. The metabolitecan be selected from acetyl L-carnitine, coenzyme Q10, glutathione, orα-lipoic acid, or other metabolites.

In preferred embodiments of the invention, the anti-inflammatoryresponse arises from one or more actions of the tissue-protective agent.Such actions can be, among other things, delaying the depletion ofadenosine triphosphate in the tissue, conserving the total adenylatepool in the tissue, buffering a decrease in the ratio of adenosinetriphosphate to free adenosine diphosphate in the tissue, delayingexhaustion of high-energy phosphates in the tissue, maintainingcell-membrane integrity in the tissue, inhibiting caspase enzymeactivity in the tissue, and reducing intracellular edema in the tissue.

In some embodiments, the tissue-protective agent crosses the blood-brainbarrier of the animal (e.g., mammal). In certain embodiments, the agentaccumulates in nerve tissue of the animal. In some embodiments, theagent reduces lactic acidosis in the tissue. The agent can also reducethe level of malondialdehyde in the tissue.

The tissue can be from any animal. Thus, it can be human tissue (ortissue of any other mammal) and can be treated in vivo or in vitro.

The tissue-protective agent can be used in methods of treating.Accordingly, the tissue-protective agent can be administeredprophylactically, therapeutically during injury, or post-injury forcontinued therapy or prophylactically against recurrence. The agent canbe administered by any suitable means, including, but not limited to,injection, orally, topically, by inhalation, or by other means. In someembodiments, the injury to be prevented or treated is related toischemia. In other embodiments, the injury is related to infection. Incertain embodiments, methods further comprise administering to theanimal an additional agent that is capable of generating nitric oxide invivo.

In accordance with the above disclosure, in another aspect, the presentinvention provides compositions for treating animal tissue subject toinjury. A preferred composition comprises (i) at least oneanti-inflammatory agent and (ii) at least one anti-apoptotic agent,where the anti-inflammatory agent is capable of inhibiting atissue-derived leukocyte chemotactic factor. In some compositions of theinvention, the anti-inflammatory agent comprises an antagonist of atissue-derived leukocyte chemotactic factor.

The identity of the anti-inflammatory agent is not limited. For example,it can be an anti-oxidant, enzyme (such as a deformylase), enzymeinhibitor, antibiotic, inhibitor of the bacterial formyl peptidechemoattractant, including but not limited to deformylase enzymes, andendogenous inhibitors of tissue-derived leukocyte chemotactic factors.The enzyme inhibitor can be, for example, L-histidine or trasylol. Incertain particular embodiments, the anti-inflammatory agent can comprisethe antagonist spinorphin, leuhistin, or in some embodiments, bothspinorphin and leuhistin. The anti-inflammatory agent of the compositioncan include the antagonist tynorphin. In some embodiments, theanti-inflammatory agent can comprise one or more agents selected fromthe group consisting of t-Boc-Phe-Leu-Phe-Leu-Phe, cyclosporine H,pentoxifylline, Carbobenzoxy-Phe-Met, and Substance P antagonistR(dextro-)PKP(dextro-) FQ(dextro-)WF(dextro-)WLL-NH₂.

According to some embodiments, the anti-inflammatory agent comprises aplurality of soluble formyl peptide receptors that can bind toformylated ligands, or a plurality of soluble formyl peptide receptorsthat can bind to non-formylated ligands. In other embodiments, theanti-inflammatory agent comprises an antibody against formyl peptidereceptors that can bind to formylated ligands, or an antibody againstformyl peptide receptors that can bind to non-formylated ligands. Incertain preferred embodiments, the anti-inflammatory comprises anantibody against a tissue-derived leukocyte chemotactic factor.

In some embodiments, the composition includes an anti-apoptotic agentcomprising a creatine analogue. The creatine analogue can becyclocreatine, a salt of cyclocreatine (e.g., cyclocreatine phosphate),or other known creatine analogues or molecular entities with the same orsimilar function to creatine analogues.

The anti-apoptotic agent of the composition can comprise a metabolite ofthe mitochondria. The metabolite can be selected from acetylL-carnitine, coenzyme Q10, glutathione, α-lipoic acid or other effectivemetabolites.

The present invention also teaches use of the compositions describedabove in treatment of mammals, wherein treatment includes bothprevention and therapy with respect to tissue or organ injury orinfection. Some aspects of the invention therefore provide a method ofusing a composition to protect or treat a first tissue of a mammal, saidfirst tissue suspected of being injured or of being susceptible toinjury, comprising providing an effective amount of the composition tosaid first tissue, wherein the composition comprises (i) ananti-inflammatory agent and (ii) an anti-apoptotic agent, and whereinthe anti-inflammatory agent is capable of inhibiting a leukocytechemotactic factor derived from a second tissue of the mammal. The firstand second tissues can be, but are not necessarily, the same tissue.Furthermore, the invention provides for use of a compound or compositionof the invention in the manufacture of a medicament, and use of thecompound or composition in a method of treating.

In yet another aspect, the invention provides a method of treatinginjured animal tissue in a subject (patient). The method generallycomprises: (a) taking a sample of a subject's tissue, which is suspectedof being damaged; (b) detecting the release of at least one peptide fromthe tissue to indicate that the tissue is in an injured state in thepatient; and (c) if the tissue is injured, administering to the subjectan effective amount of a composition comprising (i) an anti-inflammatoryagent and (ii) an anti-apoptotic agent, wherein the anti-inflammatoryagent is capable of inhibiting a tissue-derived leukocyte chemotacticfactor. Of course, due to the importance of human health, the subject isoften a human. However, in some embodiments, the subject is an animaland the method relates to veterinarian treatments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram depicting the chain of events caused by a release oftissue-derived leukocyte chemotactic factors.

FIG. 2 is a plot showing the release of high levels of chemotacticactivity from epithelial cells in response to viral infection with thelaboratory-adapted influenza virus H1N1.

FIG. 3 is a plot demonstrating that a leukocyte chemotactic factorantagonist, t-Boc-Phe-Leu-Phe-Leu-Phe (SEQ ID NO:2), inhibits fMLPchemotactic activity (fMLP is the formylmethionine peptidef-Met-Leu-Phe).

FIG. 4 is a plot showing that a leukocyte chemotactic factor antagonist,t-Boc-Phe-Leu-Phe-Leu-Phe (SEQ ID NO:2 and labeled as “Nourexin-1” or“NXin” in the figure) inhibits neutrophil chemotactic activity releasedby epithelial cells in response to viral infection with thelaboratory-adapted influenza virus H1N1.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following detailed description of certain embodiments of theinvention is provided to give the reader a better understanding ofcertain details of the invention, and is not to be understood as alimitation on the scope or subject matter of the invention.

The cell response to injury depends on the kind, severity, and durationof insult. The injury may range from mild and fully reversible to severeand lethal. Clinical effects of cell injury depend on what kind of cellis affected, its prior state of health, and what sort of adaptivemechanisms are available to it. In general, the most metabolicallyactive cells are the most susceptible to injury. For the purposes of thepresent disclosure, “injury” of cells, tissue, or organs is meant toencompass all possible sources of damage or harm to the structure orfunction of such cells, tissue, or organs, and is not to be regarded aslimiting in any way.

The most common etiologic factors involved in cell injury are asfollows:

Metabolic: such as (but not limited to) oxygen, glucose, cholesterol,and lipid profiles; vitamin A deficiency

Physical: such as (but not limited to) ischemia, reperfusion, cold,heat, trauma, dust, silica, radiation, ultraviolet, and electricity

Chemical agents: such as (but not limited to) acid and base solutions,hydrogen peroxide, pollutants, and solvents

Drugs: such as (but not limited to) acetaminophen, alcohol, narcotics,and glucocorticoids

Immunologic reactions: such as (but not limited to) allergies tosubstances and autoimmune diseases

Biologic: such as (but not limited to) viruses, bacteria, and bacterialproducts such as endotoxin, and parasites

Ischemic injury, as an example, results from interruption in blood flow.It is well-studied, particularly in heart muscle as a result of coronaryocclusion. Reversible changes are known to occur when the duration ofthe ischemia is short, e.g., 15 minutes and less. Irreversible changesoccur if ischemia persists, resulting in cellular death.

Reperfusion injury refers to damage to tissue caused when blood supplyreturns to the tissue after a period of ischemia. The absence of oxygenand nutrients from blood creates a condition in which the restoration ofcirculation results in inflammation and oxidative damage through theinduction of oxidative stress rather than restoration of normalfunction.

Cell injury can ultimately lead to cell death. Cells can die in twoways: apoptosis and necrosis, and there are distinctive differencesbetween necrotic and apoptotic cell death that can be observed andmeasured. Apoptosis is a normal, genetically controlled event whichplays a critical role in removing unwanted and potentially dangerouscells, such as tumor cells and cell infected by viruses.Morphologically, apoptosis is characterized by cell shrinkage, membraneblebbing, and chromatin condensation. The apoptotic bodies aresubsequently phagocytosed by surrounding cells or macrophages. Sincecellular contents are not released, apoptosis does not stimulate aninflammatory response. Many agents that cause necrosis can also causeapoptosis, usually at lower doses and over longer periods of time.Examples of stimuli for apoptosis include: mild ischemia, mildradiation, viral infection, and cytokines such as tumor necrosisfactor-alpha (TNF-α), lymphotoxin, and hormones such as glucocorticoids.In contrast, necrosis occurs when a cell suffers lethal injury and ischaracterized by swelling, rupturing of the cell, and inflammation (andassociated pain). The first consequence of persisted injury (ischemia,for example) is loss of oxidative phosphorylation in mitochondria andreduction in ATP production. Reduction in ATP results in failure of theATP-dependent sodium/potassium pumps and calcium pump that normallymaintain high cell potassium and low cell sodium and calcium. Beside themassive calcium influx into the cell, the drop in ATP also results inincreased glycolysis and acidosis which produces injury to lysosomalcell membranes and leakage of their powerful enzymes into cytoplasm. Theleaked lysosomal enzymes will digest the cell contents, resulting incell death. Cell necrosis in living tissue is associated with an acuteinflammatory reaction.

The balance between death by apoptosis and necrosis depends not onlyupon the intensity of the injury but also upon the level of availableintracellular ATP. A lack of ATP can cause a switch of the mode of celldeath from apoptosis to necrosis.

Many diseases are the result of hypoxia (a shortage of oxygen), oftendue to ischemia, which is an absolute or relative shortage of bloodsupply to an organ or tissue. Insufficient blood supply causes tissue tobecome hypoxic, or, if no oxygen is supplied at all, anoxic.Hypoxia/ischemia can result in inflammation (and pain) and similarly,inflammation results in vascular damage leading to hypoxia/ischemia.Both conditions are associated with apoptosis (programmed cell death)leading to tissue degeneration and loss of function. Hypoxia andischemia of major organs are also symptoms that occur in associationwith cytokine storms. A cytokine storm is a potentially fatal immunereaction comprising a positive feedback loop between cytokines andimmune cells, with highly elevated levels of various cytokines.

Injury to tissue, arising due to infection, ischemic damage, or othercauses as described above, can cause the release of mitochondrial andnon-mitochondrial proteins. These tissue-derived proteins can beformylated or non-formylated. The released proteins can be detected in asample taken from a subject, indicating that the subject has suffereddamage. One aspect of the present invention describes methods to protectagainst tissue apoptosis and to treat cytokine storms associated withimmune-system overload associated with infections (see FIG. 1), byinhibiting or blocking the release of these proteins, thereby decreasingthe likelihood of further tissue damage that could otherwise happen.

To assist in preventing and treating tissue damage caused by oxygenvariance, the present invention provides a system for causing thepreservation and prompt recovery of nerve tissue function duringischemia and following post-ischemic reperfusion. Among the tissues thatare treated are brain, ocular, and peripheral nerve tissues and nervecells within these tissues. Included in this aspect is the provision forthe administration of a tissue-protective agent before and/or afterischemia to preserve and restore nerve function of salvageable penumbratissue. Particularly, the present invention involves a system thatprovides for a three-stage treatment of (i) prevention, (ii) immediatetherapy during ischemia, and (iii) post-ischemia rehabilitation topreserve, restore, and sustain the function of nerve tissue in surgicaland non-surgical patients subjected to ischemia and reperfusion.

One feature of the present invention is to provide a potentneuro-protective or neutrophic agent, such as cyclocreatine,cyclocreatine phosphate, acetyl L-carnitine, coenzyme Q10, glutathione,or α-lipoic acid, which, when administered shortly after the incidenceof ischemia in the brain, the eye, and peripheral nerves will preservethe “salvageable tissue” surrounding the ischemic and necrotic areas andminimize devastating disability. In general, these compounds areprovided alone or in compositions in amounts suitable for achievingthese results in vivo. Administration of cyclocreatine, cyclocreatinephosphate, acetyl L-carnitine, coenzyme Q10, glutathione, and alphalipoic acid are optionally continued post-ischemia and duringreperfusion in the form of a low maintenance dose that will encourage ahealthy sustained recovery of nerve tissue function. Coenzyme Q10 is abiologically active quinone with an isoprenoid side chain, related instructure to vitamin K and vitamin E.

Because ischemic cell injury in living tissue is associated with anacute inflammatory reaction, anti-apoptotic agents, such ascyclocreatine, cyclocreatine phosphate, acetyl L-carnitine, coenzymeQ10, glutathione, or α-lipoic acid, are used in combination withtissue-derived anti-inflammatory protein(s). In case of infection,antibiotics and the inhibitors of F-Met-Leu-Phe can be added includingdeformylases. Some of the advantages of combining anti-ischemic agentswith tissue-derived anti-inflammatory agents, in accordance with someembodiments of the present invention, are as follows. The tissue-derivedanti-inflammatory agent can protect the injured tissue from damageinduced by inflammation during “early reperfusion” without affecting thenecessary healing process. The tissue-derived anti-inflammatory agentcan inhibit the release of the cytokine tumor necrosis factor-alpha fromactivated leukocytes and therefore further protect tissues against theinitiation of apoptosis and the progression of injury from reversible toirreversible. Additionally, the tissue-derived anti-inflammatory agentcan replace the immunosuppressor glucocorticoids which are known tostimulate the initiation of apoptosis in reversibly injured tissues.Further, in general, cyclocreatine, cyclocreatine phosphate, acetylL-carnitine, coenzyme Q10, glutathione, and α-lipoic acid, or otheranti-apoptotic agents with similar function, can provide and sustain theinjured cells with the necessary energy source of ATP and anti-oxidants(such as ascorbic acid) which will aid to protect the tissues againstprogressing from reversible to irreversible injury.

When cyclocreatine or cyclocreatine phosphate is selected as theanti-apoptotic agent, as in some preferred embodiments of the invention,these compounds will be stored in tissues substantially in the form ofcyclocreatine phosphate. During insult, the stored cyclocreatinephosphate can provide ATP to cells and protect the tissue from becominginjured. Creatine is phosphorylated chemically or enzymatically bycreatine kinase to generate creatine phosphate, which is well-known (TheMerck Index, No. 7315). Both creatine and creatine phosphate (also knownas phosphocreatine) can be extracted from animal tissue or synthesizedchemically. Cyclocreatine is an essentially planar cyclic analogue ofcreatine. Cyclocreatine is phosphorylated efficiently by creatine kinasein the forward reaction both in vitro and in vivo (Rowley, 1971).

Within the context of the methods of the invention and the compounds andcompositions, the compounds and compositions may be pharmaceuticals. Assuch, any pharmaceutically acceptable salt of an anti-inflammatory agentor an anti-apoptotic agent can be administered, and can be consideredwithin the term “compound” or “composition”. By “pharmaceuticallyacceptable salt”, it is meant art-recognized pharmaceutically acceptablesalts. Typically these salts are capable of being hydrolyzed underphysiological conditions. Examples of such salts include sodium,potassium, and hemisulfate. The term further is intended to includelower hydrocarbon groups capable of being hydrolyzed under physiologicalconditions, such as groups that esterify the carboxyl moiety, e.g.,methyl, ethyl and propyl. For example, salts of cyclocreatine can beadministered, rather than cyclocreatine.

Additionally, the agents of the invention can be administered either insubstantially pure form, or as part of a composition including apharmaceutically acceptable carrier, as is well-known in the art.“Pharmaceutically acceptable carrier” is intended to include substancescapable of being co-administered with the anti-inflammatory agent or ananti-apoptotic agent and that allows the active agent to perform itsintended function. Examples of such carriers include solvents,dispersion media, adjuvants, delay agents, and the like.

The administration of compositions of the invention preferably iscarried out by oral administration as a powder, or by injection,although any means of administration may be used. Injection is usuallyemployed within a fluid carrier, such as a sterile saline solution, withintravenous injection being preferred when the treatment involvespost-ischemic events. Such pharmaceutically acceptable carrier solutionstypically have an essentially neutral pH, such as the conventionallyemployed saline solution. Means for injection include, but are notlimited to, intravenous, intraperitenially, intramuscular, andintradermally. In some cases, administration may be initiated prior totissue injury and continued during and following injury.

Of course, other appropriate means of administration can be useddepending upon the particular tissue of concern and the vehicle used forits administration. Administration may include inhalation, with orwithout other means for administering compositions of the invention.Compositions can also be given topically (e.g., eye drops, lotion,cream, etc.) or transdermally.

The invention also provides for use of an anti-apoptotic agent, such ascyclocreatine, cyclocreatine phosphate, coenzyme Q10, L-carnitine,glutathione, or α-lipoic acid, alone or in combination with atissue-derived anti-inflammatory agent or agents to (a) treat(protective and therapeutic) injured tissues with the goal of protecting“normal” cells from becoming reversibly or irreversibly damaged, (b)revert the reversibly injured tissues back to normal, and (c) preventthe progression of the reversibly injured tissues toward irreversibledamage.

Among the many methods and results provided by the invention, it is tobe noted that the invention can provide a treatment for diabeticretinopathy that eliminates the destruction of photoreceptors byhigh-energy laser light and its resultant scarring.

In some embodiments, the method provides a treatment regimen associatedwith a screening test that can be employed not only pre-symptomaticallyfollowing detection, but also as an immediate treatment and continuingmaintenance program. Included in this embodiment is the provision forthe use of antagonists, blockers, and other inhibitors, includingendogenous inhibitors of tissue-specific leukocyte chemotactic factors.These antagonists can be employed as anti-inflammatory and neutrophicagents to protect cerebral, ocular and neural tissue from ischemic andinflammatory injury. Included as well is the provision for combinationsof tissue-protective antagonists that impart to the tissue atissue-specific anti-inflammatory response that delays depletion of ATP,conserves the adenylate pool in the tissue, buffers ischemic decrease inthe ratio of ATP to ADP, delays exhaustion of high-energy phosphates,maintains cell membrane integrity, reduces intercellular edema, reducesthe level of cell injury marker malondialdehyde, inhibits caspase enzymeactivity, and/or reduces apoptosis. (Malondialdehyde is the end-productof lipid peroxidation by reactive oxygen species, a measurableendpoint.) These compositions further protect the brain, spinal column,and the retinal and optic nerve tissues against ischemic injury,inflammation, and pain while reducing intracellular edema and cellinjury.

Yet another feature of the invention in embodiments is a method thatprovides the foregoing at an early stage in the pathogenesis ofage-related macular degeneration and thereby stabilizes the disease andpossibly reverses hypoxia induced oxidative stress, injury to retinalpigment epithelium and choroicapillaries and the formation of anabnormal extracellular matrix.

In another embodiment, the invention provides a treatment effective forretinal diseases secondary to ischemia, inflammation, and pain. Thepresent invention provides for early detection, monitoring, andtreatment of patients with diabetic retinopathy and age-related maculardegeneration, including early retinal damage and vascular leakage, aswell as neovascularization due to chronic and low-grade sub-clinicalinflammation. In one embodiment, the retinal-derived leukocytechemotactic factor released by ischemic and inflamed retina can be usedto develop a blood test for early diagnosis, and monitoring of diabeticretinopathy and macular degeneration and related diseases. Antagoniststo the retinal factor can be provided as anti-inflammatory agents andused to control the damaging effect induced by inflammation. Theseretinal factor antagonists can be used in combination withanti-apoptotic agents, such as creatine analogues and mitochondrialmetabolites, to maintain elevated levels of energy nucleotides andanti-oxidants during ischemia and perfusion. This treatment can reducethe chronic sub-clinical inflammation that occurs early in diabeticretinopathy and macular degeneration, inflammation that can result inretinal damage and retinal vascular leakage/neovascularization andultimately blindness.

In accordance with the present invention, the tissue-derivedanti-inflammatory agents will have the advantage of reducinginflammation-mediated tissue damage and restoring tissue functionwithout affecting the host immune system in the manner observed, forexample, in cortisone treatment.

The present invention also provides a method that includes addingendogenous inhibitors to the tissue-derived anti-inflammatory agent(s)employed to control damage induced by inflammation. That is, it providesmethods and compositions utilizing compositions having multiplebioactive agents. An anti-inflammatory cocktail could further comprisetissue factor inhibitors, endogenous inhibitors of the factor,anti-oxidants, and enzyme inhibitors that can be used in variouscombinations to specifically eliminate inflammation-mediated tissuedamage during reperfusion and restore function without affecting thehealing process that follows.

The present invention thus provides a method of achieving promptrecovery of functionality in animal, and in particular mammal, tissue.The method comprises the step of administering, preferably by injection,infusion, or orally, an effective amount of an anti-apoptotic agentprior to the onset of ischemia, or after tissue infarction, forpreserving and fully restoring tissue functionality post-ischemia. Themethod can treat maladies with anti-inflammatory agents. The malady canbe a metabolic injury, as well as ischemia and infection, such as withas injurious agents. In embodiments, the treatment reduces inflammatoryresponse in Avian influenza flu and Septic Shock Syndromes.

As a general matter, tissue-derived leukocyte chemotactic factorsrapidly released in response to tissue injury and infection have thefollowing characteristics:

LCF is an early factor which appears on the top of the inflammatorycascade before the release of other inflammatory mediators. LCFs aretypically released by injured tissues within just several minutes;

LCF serves as the initial signal in the cascade of the events that leadsto inflammation and pain. LCF not only recruits phagocytes to sites ofinjury, but also activates phagocytes and endothelial cells to release anumber of pro-inflammatory cytokines and chemokines (e.g., LECAM,ICAM-1, ELAM-1, IL-1B, IL-8, and TNF-α), oxidants, and proteolyticenzymes such as N-acetyl-B-glucosaminidase, and collagenase type IV;

LCF is not only an earlier factor, but also stimulates the release of anumber of inflammatory mediators which play a key role in the initiationand progression of the atherosclerotic lesion (IL-1, TNF-α, IL-6, MCP-1,IL-18, ICAM, VCAM, and Selectins), as well as in plaque destabilizationand rupture (MMPs, MPO, IL-18, CD40L).

Specifically, Vascular Nourin is a marker of patients with variousdegrees of atherosclerosis (i.e., patients with Coronary Artery Diseases(CAD) also referred to as Stable Angina). Currently, the enzymemyeloperoxidase (MPO, EC 1.11.1.7) is used as a marker of plaquedestabilization/rupture before patients have formed clots andexperienced myocardial ischemia (acute coronary syndromes) in the formof unstable angina or heart attack. Nourin is present much earlier thanMPO and like N-acetyl-B-glucosaminidase, Nourin will stimulate therelease of MPO from neutrophils. Unlike MPO, which is a non-specificenzyme released by activated neutrophils, Nourin is tissue-specific.Furthermore, unlike MPO levels, which are influenced by the presence ofanti-inflammatory drugs such as steroids, NSAIDS, and Statins, Nourin asa blood marker is not influenced by these anti-inflammatory drugs(steriods, NSAIDS, Statins).

One particular example of an LCF is the sequence MIINHNLAAINSH (SEQ IDNO:1). In FIG. 1, “Nourin” refers to a class of polypeptides comprisingSEQ ID NO:1 as well as all “Nourin-1” sequences disclosed in U. S.patent application publication number 2006/0063198, which is hereinincorporated by reference in its entirety.

In the tissue-derived leukocyte chemotactic factor family, it is thought(but without being limited to any particular theory) that the activepeptide has a molecular weight of about 3 KDa and is associated with alarge molecular weight carrier of 30-300 KDa. The association of the 3KDa peptide with the large carrier is an ionic, non-covalent bond. Inone example, a tissue LCF appears as a monomer in the 3 KDa band in areduced SDS-PAGE gel. However, in a non-reduced SDS-PAGE gel, thistissue LCF appears as both a monomer in the 3 KDa band and a dimer inthe 6 KDa band. MALDI analysis indicates that the 3 KDa and 6 KDaSDS-PAGE gel bands share similar peptide masses.

The LCF is stable over prolonged times when it is kept frozen (such asat a temperature of −20° C.), and as a lypholized powder kept at roomtemperature. The LCF is stable when kept in a refrigerator for 7 days.In addition, the release of the LCF is not inhibited in animal modelspretreated with dexamethasone or NSAIDS, suggesting that the pathway(s)for the formation of the LCF is independent of the arachidonic acidpathway. The release of the factor is associated with the induction ofacute and chronic inflammation. The injection of the LCF induces acuteand chronic inflammatory characterized by extensive leukocyteinfiltration, as well as fibrin and collagen deposition.

The members of the LCF family, however, differ in their isoelectricpoints, solubility in organic solvents, heat stability, and mode ofrelease. For example, the cardiac LCF has a pI of pH=7-8, while thegastric factor has a pI of pH=5.6-6.5 and the corneal derived factor hasa pI of pH=8.5. Furthermore, the gastric factor is extracted intoorganic solvents and is heat-stable, while the cardiac and cornealfactors are not extracted in organic solvents and are heat-sensitive.

Tissue-derived LCFs are a class of inflammatory mediators for leukocytesand endothelial cells. The LCFs not only stimulate endothelial cells andleukocyte chemotaxis, adhesion, and activation, but also stimulate therelease of a number of pro-inflammatory cytokines and chemokinesincluding but not limited to Interleukin-1 (IL-1), Interleukin-8 (IL-8),and tumor necrosis factor-alpha (TNF-α) by leukocytes and endothelialcells.

The present invention relates, in some aspects, to control of damageinduced by inflammation and apoptosis induced by hypoxia/ischemia,infections, and/or inflammatory mediators such as TNF-α. For infection,this invention can protect tissues against hypoxia and the prolongedimmune system overload, which kills people when infected with infectiousagents such as anthrax, Avian bird flu, and endotoxin-induced septicshock. Treatments can include any number of combinations of therapeuticcompositions comprising one or more anti-inflammatory agents effectiveto inhibit the release of tissue-derived LCFs, and in some embodiments,further comprising one or more anti-apoptotic agents.

The number of molecules capable of eliciting chemotactic responses isrelatively high, and we can distinguish primary and secondarychemotactic molecules. Formyl peptides are di-, tri-, and tetrapeptidesreleased in vivo from cells. A typical member of this group is theN-formyl-methyonyl-leucyl-phenylalanine (fMLF).

N-Formylmethionine (fMet) is an amino acid found in all living cells. Itis a derivative of the amino acid methionine. It is a modified form ofmethionine in which a formyl group has been added to a methionine'samino group. fMet is a starting residue in the synthesis of proteins inprokaryotes and, consequently, is always located at the N-terminal ofthe growing polypeptide. fMet is delivered to the ribosome (30S) mRNAcomplex by a specialized tRNA, tRNA.fMet, which has a 5′-CAU-3′anticodon that is capable of binding with the AUG start codon located onthe mRNA.

N-Formylmethionine is coded by the same codon as methionine, AUG.However, AUG is also the translation initiation codon. When the codon isused for initiation, N-formylmethionine is used instead of methionine,thereby forming the first amino acid of the nascent peptide chain. Whenthe same codon appears later in the mRNA, normal methionine is used.Many organisms use variations of this basic mechanism.

The addition of the formyl group to methionine is catalyzed by theenzyme transformylase. This modification is done after methionine hasbeen loaded onto tRNA.fMet by aminoacyl-tRNA synthetase. Methionine canbe loaded either onto tRNA.fMet or tRNA.Met. Transformylase willcatalyze the addition of the formyl group to methionine only ifmethionine has been loaded onto tRNA.fMet and not onto tRNA.Met, inwhich case the methionine will not be formylated.

Formylmethionine and non-formylmethionine ligands of the Formyl PeptideReceptors (FPR) are a class of ligands that are potent leukocytechemotactic factors rapidly released by injured tissues (within about 5minutes). The FPR competitive antagonist t-Boc-Phe-Leu-Phe-Leu-Phe (SEQID NO:2) significantly inhibits the function of LCFs on leukocytechemotaxis. Similarly, although the immunosuppressor drug Cyclosporin H(CsH) inhibits leukocyte activation induced by the FPR ligand fMLP, CsHdoes not inhibit leukocyte activation (release of histamine bybasophils) induced by C5a, IL-8, platelet activating factor, monocytechemotactic activating factor, RANTES, bryostatin 1, or phorbolmyristate, indicating that these stimulants do not appear to functionthrough the FPR (de Paulis, 1996).

“RANTES” (Regulated upon Activation, Normal T-cell Expressed, andSecreted) is an 8 KDa protein classified as a chemotactic cytokine orchemokine. RANTES is chemotactic for T cells, cosinophils, and basophilsand plays an active role in recruiting leukocytes into inflammatorysites.

Chemokines belong to a special class of cytokines. Their groups (C, CC,CXC, CX3C chemokines) represent not only structurally related moleculeswith a special arrangement of disulfide bridges, but their target cellspecificity is also diverse: CC chemokines act on monocytes (e.g.,RANTES), and CXC chemokines are neutrophil granulocyte specific (e.g.,IL-8).

FPR antagonists which inhibit neutrophil chemotaxis include, but are notlimited to:

Spinorphin (Leu-Val-Val-Tyr-Pro-Trp-Thr, SEQ ID NO:3);

Tynorphin (Val-Val-Tyr-Pro-Trp, SEQ ID NO:4);

The synthetic pentapeptide t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, SEQ ID NO:2;

The synthetic peptide t-Boc-Methionyl-Leucyl-Phenylalanine (t-Boc-MLP),SEQ ID NO:5;

Substance P antagonistR(dextro-)PKP(dextro-)FQ(dextro-)WF(dextro-)WLL-NH₂), SEQ ID NO:6;

Bile acids;

Cyclosporine H;

Pentoxifylline;

Antibodies against FPR;

Soluble FPR receptor (17 amino acid loop);

Spinorphin is an endogenous 7 amino acid peptide ofLeu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:3), which can be isolated frombovine spinal cord and inhibits both inflammation and pain. In vitro,spinorphin inhibits neutrophil chemotaxis induced by theformylmethionine peptide FMLP, and is an important endogenous regulatorfor inflammation. Spinorphin may exert its endogenous anti-inflammatoryactivity by competing with the leukocyte chemotactic factors on the FPRsites and blocking leukocyte function. Spinorphin can regulate not onlyinflammation but also pain. The proprotein of spinorphin is not known.However, spinorphin has a structure resembling that of the hemoglobulinB-subunit which exhibits opioid activity. Spinorphin and tynorphin(Val-Val-Tyr-Pro-Trp, SEQ ID NO:4) which can be isolated from monkeybrain tissue likely attenuate nociception via the inhibition ofenkephalin-degrading enzyme.

Enkephalins are possibly involved in pain-modulating mechanisms in thespinal cord and are short-lived because of rapid degradation by variousendogenous enzymes. Kyotorphin is a brain-derived protein which producesan analgesic effect by increasing the release of Met-enkephalin. Theendogenous spinorphin also increases the levels of enkephalin and reliefpain by inhibiting enkephalin-degrading enzymes. Neuropeptides such asSubstance P (Met-at C terminal), and bradykinin(H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH, SEQ ID NO:7 orK-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH, SEQ ID NO:8) induce someinflammatory responses.

Substance P is a neuropeptide with inflammatory properties. It is one ofthe tachykinins, such as bradykinin, which have contractile effects. Theprotein sequence of Substance P is RPKPQQFFGLM-NH₂ (SEQ ID NO:9). Thissequence has been shown to stimulate neutrophil chemotaxis presumably atthe Formyl Peptide Receptor (FPR). In this case, the terminal Met is atthe end of the C-terminal. The Substance P antagonist R(dextro-)PKP(dextro-)FQ(dextro-)WF(dextro-) WLL-NH₂ inhibits neutrophil-inducedchemotaxis by Substance P. It is likely that this Substance P antagonistalso inhibits LCF function. Furthermore, the migratory effect ofSubstance P on neutrophil chemotaxis is expected to be inhibited byspinorphin, which also inhibited LCF action on neutrophil chemotaxis.

Blockers of formyl peptide receptors also include soluble receptors.Clinically, soluble fragments of the receptors of the inflammatorymediators TNF-α and Interleukin-1 are used in patients with rheumatoidarthritis to inhibit the progress of disease and tissue destruction.Similarly, soluble portions of formyl peptide receptors can also beeffective in inhibiting leukocyte migration induced by endogenous formylpeptides. Furthermore, the synthetic compound t-Boc-Phe-Leu-Phe-Leu-Pheas well as the truncated 17 amino-acid loop composed of the tail regionof the FPR and having hydrophobic character, can both be effective ininhibiting leukocyte migration induced by endogenous formyl peptides.

Cyclosporine H (CsH) is an immunosuppressive drug which also acts onleukocyte FPR to inhibit their function. Addition of otheranti-inflammatory agents known in the art, such as nimbidin, can be doneto help control inflammatory diseases secondary to chemotherapytreatment and chemical toxicity, for example. Nimbidin is a mixture oftetranortriterpenes and is known to possess potent anti-inflammatory andanti-arthritic activities.

Monoclonal antibodies against LCFs can also significantly inhibitneutrophil chemotaxis and the release of, for example, IL-8 byneutrophils.

Cytokine-storm antagonists include: spinorphin, spinorphin plusleuhistin, tynorphin; t-Boc-Phe-Leu-Phe-Leu-Phe (SEQ ID NO:2), thesynthetic peptide t-Boc-Methionyl-Leucyl-Phenylalanine (t-Boc-MLP, SEQID NO:5), the immunosuppressive cyclosporine H, the FPR antagonistCarbobenzoxy-phe-met (SEQ ID NO:10), the entire FPR, or at least onesoluble receptor, the truncated 17 amino acid loop of the FPR,pentoxifylline, trasylol protease inhibitor, anti-oxidants, antibiotics,monoclonal antibodies against LCFs, monoclonal antibody against FPRwhich inhibits cell activation, cyclocreatine analogues, and saltsthereof.

The methods of the present invention can further include, in someembodiments, delivery of exogenous nitric oxide (NO). Because it can bedifficult to administer NO directly, it is preferred to administer tothe mammal an agent that is capable of generating nitric oxide in vivo.Delivery of exogenous NO, either administered directly or throughrelease by an agent that is capable of generating nitric oxide underphysiological conditions, can be a good therapeutic option in thetreatment of diseases resulting from ischemic episodes, and improve theeffectiveness of compositions of the invention. Specifically, NO canalleviate vascular spasm due to reduction of NO which occurs afterischemia. Furthermore, there may be a synergy between the vascularrelaxation effects of NO and the greater availability of ATP from anagent, since NO is required for the formation of cGMP, the cofactoressential for the kinase. This is the biochemical basis for thebeneficial effects of compounds such as nitroglycerin, an NO producer,in relieving angina attacks. Many NO providing compounds are known inthe art, and include, among others, arginine and derivatives of it,which are broken down to release NO.

The present invention can be effectively administered at a plurality oftimes associated with tissue injury, including prior to actual injury,during various stages of injury, and after injury. Specifically,compositions and methods of the invention can be used prophylacticallyto protect a mammal against the development of tissue injury, such as indisease or infection. Compositions and methods of the invention can beused after the development of tissue injury for a short period of time,such as minutes to hours. Compositions and methods of the invention canalso be used after the development of tissue injury for an extendedperiod of time, such as days to weeks, months, or even years. Finally,compositions and methods of the invention can be used after the tissueinjury has been alleviated (or there is reason to believe it has beenimproved), in an attempt to prevent the onset of a similar tissue injuryin the future. It is intended that the terms “treat” and “treatment”encompass at least all of the variations described in this paragraph, aswell as similar embodiments as will be appreciated by a skilled artisan.

The compositions and methods of the invention can be universally appliedto virtually any mammalian tissue injury due to a variety of diseasestates, infections, or other reasons. Also, the mammalian tissue can betreated even if not actually present in a mammal when treated, such asduring an organ-transplant procedure. Illustrative organ transplantsinclude lung, kidney, skin, liver, cornea, bones, and nerve cells.

The methods of compositions recited herein can be effective for bothacute and chronic inflammation. Examples of acute inflammation includestroke, peripheral arterial disease (PAD), optic nerve ischemia, pain,obesity, and stress. Examples of chronic inflammation include diabeticretinopathy and macular degeneration, diabetes, atherosclerosis, andrheumatoid arthritis. The methods of compositions recited herein canalso be effective for infections, such as septic shock and anthrax;and/or tissue states associated with lethal cytokine storms such asAvian Influenza.

The compounds, compositions, and methods of the present invention haveapplicability in treatment of many tissue injuries and biochemicalstates within tissues and cells. Some non-limiting examples include:atherosclerosis, rheumatoid arthritis, brain ischemia and trauma, spinalcord ischemia and trauma, aortic cross clamping leading to spinal cordischemia, kidney ischemia and trauma, liver ischemia and trauma, lungischemia and trauma, muscle ischemia (e.g., PAD), inflammation, trauma,and atrophy, skin ischemia and trauma, ocular ischemia, degeneration,and inflammation, nervous system ischemia, degeneration, andinflammation, digestive system (e.g., esophagus, stomach, intestine,colon) ischemia, degeneration, and inflammation, spleen and pancreasischemia, degeneration, and inflammation, ear, mouth, and nose ischemia,degeneration, and inflammation, bBone marrow ischemia, degeneration, andinflammation, sexual organs ischemia, degeneration, and inflammation,dental tissues ischemia, degeneration, and inflammation.

Therapeutic applications of inhibitors of vascular LCF, to prevent andtreat atherosclerosis, include, but are not limited to, the following:reducing the lesion size, reducing the formation of new plaques,reducing the size of plaques that already exist, stabilizing plaques andmake them less prone to rupturing and forming clots, altering cellularcomposition of atherosclerotic lesions, inhibiting recruitment ofphagocytes to vessels and accordingly inhibiting the foam cell formationof macrophages, limiting the progression of the atherosclerotic lesions,and reducing the progression of the atherosclerotic lesion.

Diabetes results in vascular damage and reduction of sufficient bloodflow to tissues, leading to the development of a number ofischemia-related diseases such as diabetic retinopathy, peripheralartery disease, optic nerve ischemia, and cerebrovascular diseases.Injury to the vasculature also results in ischemia of nerve tissue andthe development of many neuro diseases. Hydrogen peroxide as sometimesreleased as an injurious agent to tissues leading to the release of LCFsfrom tissues such as corneal endothelium and epithelium, retina, and theheart. The present invention can protect the vasculatures from damagesecondary to diabetes, as well as other diseases that can also inducevascular injury such as in the case of anthrax infection, chemotherapyin cancer patients, patients experiencing various burns, etc. Ischemiaand inflammation play a key role not only in stroke (brain ischemia) butalso in the early pathogenesis of diabetic retinopathy and age-relatedmacular degeneration. Diabetic retinopathy is a leading cause ofblindness worldwide.

With regard to cerebral and peripheral nerve tissue, it is understoodthat stroke is one of the leading causes of death and long-termdisability in the United States. There are two forms of stroke:ischemic, characterized by blockage of a blood vessel supplying thebrain and the resultant cessation or reduction of blood flow; andhemorrhagic, characterized by bleeding into or around the brain. Thepresent invention can treat stroke of the ischemic and hemorrhagic type.

It is known that the arterial wall is one of the most poorly oxygenatedtissues in the mammalian body. In larger arteries, the central part ofthe arterial wall is very poorly supplied with oxygen and othernutrients. Using an atherosclerotic rabbit model, it was reported thathypoxia, low ATP content, high lactate content, and low glucose contentwere shown in the atherosclerotic lesions as well as in the media(Bjornheden et al., 1999). These studies suggest that hypoxia and energydeficiency may be major contributors to the generation of centralnecrosis in atherosclerotic lesions. Hypoxia may stimulate the releaseof vascular LCF and contribute to the inflammatory response in theplaque. Vascular LCF may also stimulate the release of a number ofinflammatory mediators which are involved in the development ofatherosclerotic lesions, including cytokines such as TNF-α, IL-1, IL-6,and IL-8; adhesion molecules such as Selectins, MPO, and MCP-1, whichrecruit monocytes and lymphocytes and promotes plaque destabilization.Vascular LCF can play a crucial role in the initiation and propagationof atherosclerosis.

A number of studies support an important role of diet and inflammationin the risk of a variety of chronic diseases, including diabetes,atherosclerosis, vascular diseases such as peripheral artery disease,and cerebrovascular diseases and overall mortality (see, for example,Esposito K, Diet and inflammation: a link to metabolic andcardiovascular diseases, Euro Heart J, 27(1):15-20, 2005). Evidence isavailable indicating that the generation of a pro-inflammatory milieumight be one mechanism through which unhealthy diets are linked tometabolic and vascular diseases. Understanding the link between diet andinflammation holds the premise to elucidate the mechanisms by whichdietary patterns improve cardiovascular health.

More evidence is building in support of the idea that obesity causesinflammation and that chronic inflammation is a major cause of manydegenerative diseases and accelerates aging. Published studies reportedthat circulating monocytes and lymphocytes exist in a pro-inflammatorystate in obese persons known to be at increased risk of developingdiabetes, vascular disease, stroke, etc. Pro-inflammatory factors weresignificantly higher in blood samples from obese subjects than theaverage weight and that the index of insulin resistance in the obesesubjects was nearly three times higher than that of the normal subjects.The hyper inflammatory status interferes with insulin signaling whichresults in insulin resistance and ultimately diabetes. In diabeticpatients, there is an increased circulating levels of cytokines such asTNF-α, Interleukin-6, Interleukin-18, and C-reactive protein (CRP—amarker of endothelial dysfunction) (Ceriello A, Evidence for anindependent and cumulative effect of postprandial hypertriglyceridaemiaand hyperglycaemia on endothelial dysfunction and oxidative stressgeneration. Circulation, 2002, 106:1211-1218). Changing diets and losingbody weight, however, was shown to significantly reduce the levels ofcytokines and insulin resistance.

Macronutrient intake may produce oxidative stress and inflammatoryresponses. The raised flux of nutrients in the post-prandial state isassociated with an increase in circulating levels of pro-inflammatorycytokines, recruitment of neutrophils, and oxidative stress. Diet reachin natural anti-oxidants (fruits, vegetables), the elimination of trans-and saturated fatty acids intake, and increasing the consumption ofomega-3 fatty acids were shown to be associated with reducedinflammatory status (Esposito K, Diet and inflammation: a link tometabolic and cardiovascular diseases, Euro Heart J, 27(1):15-20, 2005).Interestingly, emotional states have a large influence on bodyinflammation. People who are prone to anger, hostility, and depressivesymptoms respond to stress with increased production of the stresshormone norepinorphine. The increase in this stress hormone activatesthe inflammatory arm of the immune system resulting in chronic,low-grade inflammation. Therefore, it was recommended that losing excessweight and lower daily stress will lower the level of body-wideinflammation and the person will live longer.

We have shown that Nourin is a key early inflammatory mediator releasedby various tissues in response to oxidative injury, nutritional factorsincluding vitamin A deficiency, and infections (viral and bacterial). Incase of obese individuals, Nourin is similarly released in response toinjury induced by oxidized cholesterol namely oxidized LDL. Nourin alsoactivates leukocytes and vascular endothelial cells to release a numberof chemokines and cytokines including adhesion molecules, IL-1, IL-8,IL-6, and TNF-α. As early initial signal in the inflammatory cascade,Nourin can be used both as a “disease marker” and “therapeutic target”.

Adipose tissues acquire an influx of new blood vessels to support theincreased nutritional demands. Adipocytes release angiogenic factors toinduce this process. This means that the variety of pro- and competentinflammatory cytokines that have been stimulated by the early markernourin can have ready access to the adipose tissue and exert theirdeleterious effects on the host. If nourin significantly mobilizes VEGFfor example, this would be an additional reason to devise inhibitors fornourin.

MCP-1 is involved in the initiation of the fatty streak by recruitingmonocytes and lymphocytes, promotes plaque destabilization. MCP-1 is amember of the chemokines (C-C) family with molecular weight of 10 KDa(102 aa). It is not induced, however, by ischemia as Nourin does and itis induced only after postischemia during reperfusion and, accordingly,it is considered as a late chemoattractant after C5a. Treatment of humanendothelial cells with oxidized LDL also induced MCP-1 secretion. Nourinwill be released earlier than MCP-1 in response to both hypoxic injuryand oxidized LDL treatment.

Diagnostically, the blood levels of Nourin alone and in combination withother inflammatory mediators such as IL-1, IL-8, IL-6, TNF-α, and CRPcould serve as an easy “early warning” for inflammation in obese andstressed individuals. Nourin is much earlier than CRP as a marker ofendothelial dysfunction and disease development. Unlike CRP which is anon-specific inflammatory mediator released by liver in response toinjury, Nourin is a tissue specific marker. Therapeutically,anti-inflammatory drugs such as Nourin antagonists and anti-apoptoticdrugs such as cyclocreatine will be useful in controlling inflammationassociated with obesity and stress and accordingly prevent thedevelopment of a number chronic diseases including diabetes,cardiovascular, and cerebrovascular disease.

In general, a cytokine storm is induced by viral infection (e.g.,seasonal and Avian influenza flu), gram-negative bacterial infection(endotoxin), and also in patients infected with virus and thengram-negative bacterial infection due to a compromised immune system.The systemic expression of a healthy immune system results in therelease of many pro-inflammatory mediators including chemokines,cytokines, oxygen free radicals, digestive enzymes, and coagulationfactors and can lead to organ failure and death. Key pro-inflammatorycytokines are TNF-α, Interleukin-1 (IL-1), InterLeukin-8 (IL-8), andInterleukin-6 (IL-6), as well as anti-inflammatory cytokines such asInterleukin-10 and Interleukin-1 receptor antagonist.

The tissue-derived Nourin released shortly after Avian viral infectionor the lethal bacterial infection such as E. coli will be one of the keyinflammatory mediators that activate resident macrophages and recruitleukocytes to release high levels of adhesion molecules, proteolyticenzymes, oxidants, chemokines and cytokines including IL-1, IL-8, IL-6,and TNF-α. The early release of Nourin by viral and bacterial infectedtissues and its key role in evoking the observed cytokine storms will,therefore, be the basis for developing therapeutic products aimed atcombating excessive host inflammatory response which kills patientsinfected with for example the Avian influenza and E. coli. Therapeuticproducts aimed at reducing the levels of the tissue-derived Nourin willalso inhibit the levels of several pro-inflammatory mediators includingTNF-α and, therefore, additionally protect tissues from TNF-α inducedapoptosis.

LCFs stimulate local tissues and immune cells to release key chemokinesand cytokines of the cytokine storms which result in organ failure anddeath. LCF antagonists can play a key role in controlling hostinflammatory reactions caused by the Avian flu virus which forces thebody's immune system into overdrive, attacking internal organs. In someembodiments, LCF antagonists are not affected by changes in viralstrains or virus mutations and specifically inhibit the cytokine stormswithout being subjected to viral resistance (a significant concern forcurrent viral medications). In preferred embodiments, the treatmentcomprising LCF antagonists is specific to inhibit LCF-induced cytokineswithout affecting the host defense immune system.

Anthrax is a lethal gram-positive bacterium that lacks endotoxin.Despite the long history of B anthracic as a human and animal pathogenand its notoriety as an agent of biological warfare, exactly how anthraxkills the host is unclear (Prince, 2003). Major pathologicalobservations include vasculitis (vascular inflammation of small andlarge vessels), vascular apoptosis, edema, endotoxin shock-like cytokinestorms, hypoxia, apoptosis and necrosis of major organs, organ failure,hemorrhage, and death of the mammal. Bacillus anthracis, the causativeagent for anthrax, produces three polypeptides that comprise anthraxtoxin. These are protein antigen (PA), lethal factor (LF), and edemafactor (EF). The lethal factor is a matrix metalloproteinase enzymewhile the edema factor is an adenylate cyclase enzyme.

In a pathological study of human inhalational anthrax after a largeoutbreak in Sverdlovsk, Russia, hematogenous spread of Bacillusanthracis was found to be associated with capillary and vascular lesionsthat consist of fibrin deposition and various amount of neutrophicinfiltrate surrounding the vessel wall (Grinberg, 2001). The capillariesand vasculitis weakened the vessel wall and produced high- andlow-pressure hemorrhages (Grinberg, 2001). Injecting corneal LCF intorabbit anterior chamber resulted in neutrophil and fibrin accumulation,as well as corneal edema within two hours (Elgebaly, 1994). It hasfurther been demonstrated in vitro that neutrophils induce endothelialdamage and denude the membrane leading to edema (Elgebaly, 1984).

In some embodiments of the present invention, a composition comprisingcyclocreatine can be effective to moderate the edema associated withanthrax. Anthrax edema toxin (ET) consists of a protective antigen (PA)and an adenylate cyclase edema factor (EF), which is a Ca++/Calmodulinenzyme. The massive production of cyclic AMP (cAMP) from B. anthracisinfection is associated with prominent inflammation and swelling oftissues. An analogous effect is produced by choleratoxin, e.g., cAMP isa ubiquitous cofactor for many protein kinases, some of which areinvolved in rapid changes in capillary permeability. Such enzymesrequire ATP to function. The creatine kinase pathway has a critical rolein the efficient mobilization and use of ATP for energy. The naturalsubstrate, creatine (Cr), exchanges high energy phosphate (˜P) with ADPin the kinase reaction. However, an unnatural substrate, cyclocreatine(CCr) can also participate in the kinase reaction. The transfer of ˜Pfrom CCrP to ADP is at a significantly lower rate than the comparablereaction with CrP.

Therefore, it is hypothesized (without being limited to any particulartheory) that CCr may compete for available ATP with kinase enzymesdedicated to changes in vascular permeability which require cAMP foractivity. In other words, the EF (adenylate cyclase) that produces cAMPwould encounter a reduced pool of ATP in the presence of a givenconcentration of CCr. The latter has been shown to be relativelyinnocuous in mammalian systems. Significant roles for prostaglandins andhistamines are known in the inflammatory cascade. With respect to ET ithas recently been shown that the use of PGE synthase inhibitors(Celecoxib) and histamine antagonists (Cromolyn) has ameliorated theedematous effects of ET (Tessier et al., 2007). Because these eventsoccur distal to the generation of cAMP, the administration of CCrtogether with requisite antibiotics should be considered in thetreatment of anthrax infection, especially the inhalational kind.

It is known that L-histidine, at pharmacological levels (such as about10 mM), can inhibit the matrix metalloproteinases (MMP) of the cytokineAutotaxin. The latter is a tumor cell-associated moiety that hydrolyzesa phospholipids substrate to yield a potent cytokine that has motogenicand mitogenic properties. The amino acid has also been shown to inhibita gelatinase activity (Clair, 2005). L-histidine is quite specific andcan be an effective agent in the compositions of the invention whenapplied to treat anthrax infection. In preferred embodiments, anthrax istreated with an anti-inflammatory agent comprising LCF to inhibit earlyevents such as vasculitis and vascular damage, an anti-apoptotic agentto protect tissue from late events such as hypoxia and apoptosis (andresultant organ failure), and L-histidine to protect against thedamaging effect of MMP.

The present invention can be used for mammalian patients who willundergo nerve-related surgery, such as aneurysms, tumor, intracerebralhemorrhage surgery, and other similar procedures (e.g., neuromusculardiseases), as well as patients who will undergo “non-nerve” relatedsurgery that is capable of causing ischemia of the nervous system. Thesystem includes the administration of effective agents (as describedabove) before, during, and after ischemia to preserve and restore nervefunction before and during reperfusion.

The present invention can be used during many treatment stages mentionedabove. It can be used prophylactically prior to ischemia to protectagainst stroke, ocular, and peripheral nerve damage in high-riskpatients including aging populations, diabetic patients, patients withvascular diseases, and patients with prior transient ischemic attack(TIA). It can be employed prior to nerve-related and “non-nerve” surgeryto protect against ischemic injury and neurologic complications. Inaddition, it can be used immediately after cerebral ischemic stroke toprotect surrounding areas against tissue injury and loss of function,immediately after hemorrhagic stroke to protect surrounding areasagainst tissue injury and loss of function, immediately after ischemiaof peripheral nerves to protect surrounding areas against tissue injuryand loss of function, or immediately after head trauma to protectsurrounding areas against ischemic and necrotic tissue injury and lossof function. It is also applicable immediately after spinal cord traumaor peripheral nerve trauma to protect surrounding areas against ischemicand necrotic tissue injury and loss of function. Finally, it can beemployed as a maintenance treatment post-ischemia and post-reperfusionof nerve tissue to assure a healthy recovery and continued nerve tissuefunction.

Administration of an anti-apoptotic agent shortly prior to ischemia (forexample, four days or less prior to an ischemic condition) can beeffective to restore post-ischemic nerve function. Such administrationcan have application to high-risk patients to protect against ischemicdamage and reperfusion, as well as to presurgical patients to preventloss of nerve function associated with post-ischemic reperfusion.

Unlike tPA therapy, the treatments of the invention can be given topatients experiencing stroke attack as a result of hemorrhage orblockage to protect areas at risk and reduce disability. Additionally,it can be immediately administered to patients experiencing ischemicstroke upon presentation to an emergency room, even beyond thethree-hour therapeutic time window that often leads to treatmentdisqualification with tPA. Unlike tPA therapy, the treatments of thepresent invention would not be expected to have neurotoxic or vasoactiveside effects, alter the blood-brain barrier, or pose a risk ofhemorrhage.

The effective amount of the at least one agent to be administered in theinvention will depend on a number of factors associated with the type ofinjury, the patient, the properties of the agent or agents, andenvironmental conditions at the time of treatment. For purposes ofillustration, effective amounts of preferred anti-apoptotic agents willbe described. Sterile saline solutions containing substantially morethan one percent and typically more than three percent by weight ofcyclocreatine can be effective within three days of surgery, althoughsomewhat lower percent levels of cyclocreatine may be effective whenadministered within five to seven days prior to surgery. The dosageadministered may be as low as about 1 gram per 70 kilograms of bodyweight but typically is greater than about 5 g/70 kg. For cyclocreatineand cyclocreatine phosphate, a saturated solution of 0.1%-5% (or higher)of cyclocreatine and/or cyclocreatine phosphate can be prepared insaline or any other physiologic buffer.

Acetyl L-carnitine can be administered orally or as injection as low as2 grams per 70 kilograms of body weight. Higher amount of acetylL-carnitine can be used. Similarly, glutathione and α-lipoic acid can beadministered each orally or as injection as low as 2 grams per 70kilograms of body weight. Higher amount of α-lipoic acid can be used.

EXAMPLES

The present invention will now be further characterized and understoodby reference to the following non-limiting examples. The followingparagraphs describe experimental procedures that demonstrate thebeneficial effects of the administration of anti-inflammatory agentsalone and in combination with anti-apoptotic agents in protectingtissues from injury induced by ischemia, inflammation, and pain. Forthese studies, animal models and cell culture models of tissue ischemiaand inflammation that represent ischemia and infection were used.

Example 1 Release of Leukocyte Chemotactic Factors by Ischemic SpinalCord Tissue

Three pigs were sacrificed and the spinal cord were immediately removed,cut, and incubated in Hank's Balance Salt Solution (HBSS) at roomtemperature (1 gm spinal cord/2 ml HBSS). After 5, 10, 20, 40, 60, and240 minutes, 100 microliter (ul) aliquots of supernatant solutions werecollected and tested for the level of neutrophil chemotactic activityusing human peripheral neutrophils as indicator cells. Samples collectedfrom the 2 hour incubation were also processed on the size exclusionHPLC using the 1-300 KDa column. Modified Boyden chambers were used totest for the chemotactic activity in aliquot samples as undiluted, aswell as diluted 1:5, and 1:25 in HBSS. The synthetic f-Met-Leu-Phe (10⁻⁹Molar) was used as the positive control for 100% response, while HBSSwas used as the negative control for random migration. Neutrophilmigration was reported as chemotactic index of cell density (O.D. Units)using a LKB laser densitometer.

High levels of neutrophil chemotactic activity (75-95% of f-MLP) weredetected as early as 5 minutes after ischemia. Activity continued to bedetected (50-110% of f-MLP) for the additional four hour incubation.When culture supernatant solutions were fractionated using sizeexclusion HPLC (1-300 separation), high levels of chemotactic activity(50-100% f-MLP) was detected in fractions below 5 KDa. Activity wasdetected in undiluted, as well as fractions diluted 1:5 and 1:25. Thisfinding demonstrates the release of a potent low-molecular-weightchemotactic factor from ischemic spinal cord tissue minutes afterischemia and that the release of the factor was sustained for theduration of ischemia.

Example 2 Alcohol-Induced Inhibitors of Leukocyte Chemotactic Factors

Studies have shown the association between acute and chronic ethanolintoxication and lowered resistance to infection in these patients(Feliu, 1977). Impairment of neutrophil chemotaxis due to the presenceof serum inhibitors was suggested as a major mechanism for the increasedsusceptibility to infection (VanEpps, 1975). To date, tissue(s)releasing these serum inhibitors has not yet been identified.

Because the mucosal side of the gastric tissue is the first to beexposed to ingested ethanol and is exposed to the highest concentrationsfor a length of time, we tested the capability of gastric mucosa exposedto ethanol to release inhibitors of neutrophil chemotaxis. The mucosalsurfaces of rabbit stomachs were incubated for 60 minutes with 0.01%ethanol (V/V) while the serosal sides were incubated with buffer.Results indicate that gastric tissue exposed to ethanol releasedinhibitors for neutrophil chemotaxis. Control non-ethanol treatedgastric mucosal tissue released high levels of the gastric neutrophilchemotactic factor (Elgebaly, 1990). An average suppression ofneutrophil chemotaxis compared to control non-ethanol treated gastricmucosal samples was 51%. There was no detectable level of ethanol in theserosal samples. When the serosal solutions of alcohol-treated stomachswere diluted 1:3 and 1:9 in PBS, the recovered chemotactic activityremained low in both dilutions indicating that the observed reduction inactivity is not due to a desensitization effect on neutrophils butrather the presence of inhibitors.

Interestingly, when the serosal solutions of alcohol-treated mucosa werefractionated using an amicon Ultrafiltration membrane of molecularweight cut off 100 KDa, the high (above 100 KDa) and low (below 100 KDa)fraction inhibited neutrophil chemotaxis induced by C5a. The high andlow molecular weight inhibitor reduced C5a-induced neutrophil chemotaxisby 54% and 53%, respectively. The nature of the high and low molecularweight tissue-derived LCF inhibitors could be endogenous competitiveantagonists of C5a on neutrophil receptors, a proteolytic enzyme whichnon-specifically deactivates LCF and reduce their activity, or a“peptide deformylase” which deformylate N-formylmethionine peptides andreduce their activity (Nguyen, 2003).

The generation of these endogenous inhibitors of inflammation by localtissue indicates the capability of injured tissue to release both pro-and anti-inflammatory factors in response to various treatments. Thegeneration of these gastric-derived immunosuppressor following alcoholconsumption might increase the susceptibility of individuals to variousinfections including the Human Immunodeficiency Virus (HIV). Resultspresented in this Example 2 support a role for alcohol as a cofactor inimmunosuppressive disorders. Therefore, the present invention providesan opportunity to further identify, develop, and incorporate theseendogenous inhibitors into the tissue specific anti-inflammatorycocktails employed to control damage induced by inflammation.

Example 3 Leukocyte Chemotactic Factors Released by Epithelial CellsInfected with H1N1 Virus

We determined whether epithelial cells (Madin-Darby canine kidneycells-MDCK) grown in culture release LCFs in response to injury inducedby laboratory-adapted influenza H1N1 virus. For these studies, MDCKcells were infected with the laboratory influenza virus H1N1 (PR8) for1, 3, 6, 12, and 24 hours. Control cells were incubated with culturemedia only. At the various time points (1-24 hours), supernatantsolutions were collected and tested for the presence and levels ofchemotactic activity using standard modified chemotaxis chamber as afunctional assay for chemotactic factors. Neutrophils isolated fromhuman peripheral blood were used as the migratory cells.

As described in FIG. 2, significant chemotactic activity is detected insupernatant solutions collected from H1N1 infected cells compared tocontrol cells grown in culture without the virus. High levels ofchemotactic activity were detected as early as 6 hours after viralinfection and remained high for the remaining 24 hours of incubation.Control MDCK cells incubated with culture media released low levels ofthe chemotactic factors. This data suggests the ability of epithelialcells to quickly release (within first 6 hours) high levels ofchemotactic factor (referred to as Nourin in FIG. 2) in response toinfluenza viral injury.

Results described in FIG. 3 and FIG. 4 indicate that an LCF antagonistof MIINHNLAAINSH (SEQ ID NO:1) significantly inhibited neutrophilchemotaxis induced by viral chemotactic factor (30%-100%) atconcentrations ranging from about 10⁻⁵ to 10⁻³ Molar. The LCF antagonistis t-Boc-Phe-Leu-Phe-Leu-Phe (SEQ ID NO:2). These results suggest thatat least one of the chemotactic factors released by epithelial cells inresponse to H1N1 viral infection is MIINHNLAAINSH. Supernatant solutionsused in this study were obtained from cultured MDCK cells incubated withthe influenza virus H1N1 for 6 hours. FIG. 3 demonstrates that thisleukocyte chemotactic factor antagonist inhibits fMLP chemotacticactivity (fMLP is the formylmethionine peptide f-Met-Leu-Phe). FIG. 4shows that this antagonist inhibits neutrophil chemotactic activityreleased by epithelial cells in response to viral infection with theseasonal influenza virus H1N1.

Example 4 Leukocyte Chemotactic Factors Released by Various Tissues inResponse to Endotoxin Treatments

We demonstrated the release of the 3 KDa LCF Nourin by a number oftissues in response to endotoxin treatments both in vitro and in vivo.In vitro, high levels of the 3 KDa Nourin was isolated from culturedhuman bladder fibroblasts treated with endotoxin for 6 hours. Culturesupernatant solutions were collected and the low molecular weight Nourin(3 KDa) was isolated using size exclusion high performance liquidchromatography (HPLC). In vivo, rabbit eyes were injected intravitreallywith 100 ng E. coli endotoxin. Control eyes were injectedintramuscularly with 1 ml cod liver oil. After 24 hours, rabbits wereeuthanized and the aqueous humor (AH) and corneas were removed. Theepithelial surfaces of isolated corneas were incubated with 1 ml culturemedium (MEM) at room temperature for 1, 4, and 6 hours. Cornealsupernatant solutions and AH samples were assayed for the presence ofneutrophil chemotactic activity using modified Boyden Chambers.

There were detectable levels of protein content (33.4±3.4 mg/ml),neutrophil chemotactic activity (61±15% fMLP) and neutrophil cell count(17,800±2,400 cells/nm³ in AH samples of endotoxin treated eyes. A highlevel of chemotactic activity was also detected in corneal supernatantsafter 1 hr of incubation with release persisting for 4 hr, followed by adecline by 6 hr, presumably due to enzymatic deactivation. The 3 KDacornea-derived Nourin was identified (93±1.1% f-MLP) in the cornealsupernatant solutions. Control eyes, on the other hand, did not show anelevation of cell count or protein content in AH samples. Similarly,there was no release of chemotactic factors from isolated corneasincubated in buffer solution. In conclusion, the 3 KDa Nourin isdetected in AH and released by corneal tissues of an endotoxin-Uveitismodel of rabbit.

Example 5 Leukocyte Chemotactic Factors Released by Vascular Tissues inResponse to Ischemic Injury and Shear Stress (Pressure)

Isolated bovine coronary artery pieces: The release of neutrophilchemotactic factor from vascular tissues was demonstrated using isolatedbovine coronary arteries. Coronary arteries were isolated from freshbovine hearts then cut into pieces and incubated for 1, 2, 3, and 4hours at room temperature. High levels of chemotactic activity (2-3 foldof the positive control FMLP) was released by the first hour ofincubation and continued on for additional three hours (Elgebaly, 1987).

Isolated Canine Vein Grafts: Significant release of neutrophilchemotactic factor was also detected in extended vein grafts of dogs.For these studies, leg veins were isolated and extended under pressure(300 mmHg) for 15 minutes then the inner surfaced allowed to incubatewith buffer for an additional 45 minutes. High levels of chemotacticactivity (5-7 fold) were detected in the inner buffer solutions isolatedfrom the extended vein grafts compared to the in situ control veinswhere the blood was removed and replaced with buffer for 1 hour withoutany extension (Elgebaly, 1990).

Isolated Human Vein Grafts: Patients' leg veins were removed from fivepatients scheduled for coronary bypass surgery. The samples for eachpatient were distended for 10 minutes at 300 mm Hg pressure andincubated in buffer (HBSS) for 1 hour at room temperature. Undistendedvein grafts were incubated at room temperature in HBSS for one hour andthen the solutions were tested for chemotactic activity.

Chemotaxis Assay: The chemotactic activity of vein samples was testedusing standard chemotaxis assay. Briefly, neutrophils were isolated fromhuman peripheral blood and labeled by fluorescence dye. These labeledneutrophils were used as migratory cells and placed on the top chamberof the 96 well chemotaxis plates. The vein samples were placed at thebottom wells of the plate. A filter was placed in between and theability of the tested samples to stimulate the migration of neutrophilsacross the membrane was determined by incubating the chamber at 37° C.for 1 hour. The standard synthetic chemoattractant f-Met-Leu-Phe (f-MLP)was used as the positive control for 100% chemotactic response. Hank'sBalanced Salt Solution (HBSS) was the negative control for randommigration. Neutrophil migration was reported as the number of labeledneutrophils detected at the bottom wells which crossed the membranefilter in response to the test solutions. The samples were tested intriplicate wells.

Inhibition of the human vascular Nourin by the N-formylmethionyl blockert-Boc-PLPLP: t-Boc PLPLP is a FPR antagonist and was tested for itsability to inhibit Nourin activity on neutrophil migration. In thechemotaxis chamber, t-Boc-PLPLP was used at a final concentrations of10⁻⁵ Molar. Results indicated that that t-Boc-PLPLP at 10⁻⁵ M inhibitedup to 60% of neutrophil chemotaxis induced by vascular Nourin. In onehuman sample, t-Boc-PLPLP inhibited 100% of neutrophil chemotaxis.Similarly, the FPR antagonist Spinorphin inhibited neutrophil chemotaxisstimulated by Nourin.

Results of this human study support that a potent chemotactic factor israpidly released within 10 minutes by vascular tissues undernon-physiological conditions including ischemia and pressure extension.Vascular Nourin, similar to epithelial Nourin, is inhibited by FPRantagonists such as t-Boc-PLPLP.

Example 6 Tissue Protection by Cyclocreatine and Reduction of Apoptosis

Cyclocreatine (25 gm in 500 ml saline) was injected intravenously into adog one hour before the induction of global warm ischemia. Control dogsreceived saline. All dogs underwent one hour of global warm ischemia bycross clamping the aorta. After the one hour of warm ischemia, heartswere removed, perfused for an additional four hours with buffercontaining 1% cyclocreatine (20 gm in 2 liters), and then placed on aworking Longerdorff to measure cardiac function. After the initiation ofthe aortic cross clamping, cyclocreatine-treated heart continued to beatfor 9 minutes during warm ischemia, while the control heart stoppedbeating after 2 minutes. Similarly, myocardial pH was 7.04 in thecyclocreatine-treated heart compared to pH 6.00 in control heart whenmeasured 6 minutes after the induction of warm ischemia.

Biochemical and functional analyses demonstrating the cardioprotectionby cyclocreatine treatment are as follows: three-fold increase ofmyocardial ATP as tissue energy metabolism in cyclocreatine-treatedheart compared to controls; significant reduction in apoptosis incyclocreatine-treated heart compared to controls as measured by caspaseenzyme activity; reduced intracellular edema compared to control asmeasured by MRI; reduced level of the cell injury marker malondialdehydecompared to controls; reduced myocardial tissue lactic acidosis comparedto control (per MRI); reduced level of the cardiac-derived leukocytefactor; reduced inflammation/pain and injury induced by inflammation;reduced TNF-α released by monocytes and therefore reduced apoptosis.

The cyclocreatine-treated heart continued to show strong contractilitythroughout the one-hour analysis on the Longerdorff working heart, whilecontrol hearts showed strong contractility only during the first 15-20minutes then the contractility declined. The results indicated areduction of the caspase enzyme activities in the cyclocreatine dog(26-76% reduction of baseline) compared to the significant stimulationobserved in control dogs (1.44-3.86-fold increase over baseline).Interestingly, the significant reduction of caspase activities indicatesthat the enzymes may be present more in the “inactive proenzyme” forms.Cyclocreatine treatment may stimulate the anti-apoptotic members of thefamily such as Bc1-2 and/or Bc1-x, or inhibit the pro-apoptotic membersof the family such as Bak, Bax, and Bim.

In general, when cells are injured, the anti-apoptotic Bc1-2 and/orBc1-x are lost from the mitochondrial membrane and are replaced by thepro-apoptotic Bak, Bax, and Bim. Furthermore, when BC1-2/Bc1-x levelsdecrease, the permeability of the mitochondrial membrane increases, andseveral proteins that can activate the caspase cascade leak out. One ofthese proteins is cytochrome C, well-known for its role in myocardialrespiration. In the cytosol, cytochrome C binds to a protein calledApaf-1 (apoptosis activating factor-1), and the complex activatescaspase enzymes.

Because apoptosis plays a very important role in the pathogenesis ofischemia-induced tissue failure and loss of function, reducing caspasesactivities by cyclocreatine in this example helps prevent and treatorgan injury in nerve and ocular tissues. Further, because ATP depletionis a major event in ischemic stroke, peripheral nerve ischemia, andretinal damage, preservation of the energy source in such organs cansignificantly protect the nerve tissues (brain, spinal column, retinaltissues, and optic nerves) from ischemic injury and restorefunctionality.

Example 7 Role of Leukocyte Chemotactic Factors in the Development ofCytokine Storms

It is known in the art that serum TNF-α concentrations in excess of 1ng/ml in a patient are frequently predictive of a lethal outcome. Asdescribed in Table 1, the tissue-derived LCF Nourin incubated with humanmonocytes for only 4 hours stimulated the release of significantly highlevels of the cytokine storms key inflammatory mediators including TNF-α(400 pg/ml), IL-1β (400 pg/ml), and IL-8 (12,000 ng/ml). Monocytesincubated with control media released significantly less chemokines andcytokines. These data further supports a role of the endogenous Nourinas an “early” potent mediator in the pathogenesis of cytokine stormsdocumented to occur secondary to complications of Avian flu infection.

Studies by Tumpey and co-workers (Tumpey et al., 2005) demonstrated thedetection of 350 pg/ml TNF-α in lung homogenates of mice infected withthe highly pathogenic 1918 influenza virus for 5 days. It is believedthat epithelial-derived Nourin released by lung tissue shortly afterviral infection is one of the key inflammatory mediators which activateresident lung macrophages and recruited monocytes to release high levelschemokines and cytokines including TNF-α. The early release of Nourin byinfluenza infected lungs and its key role in evoking the observedcytokine storms can, therefore, be the basis for developing therapeuticproducts aimed at combating excessive host inflammatory response whichkills patients infected by the Avian influenza. Therapeutic compositionsof the invention that reduce the levels of the tissue-derived Nourinwill also inhibit the levels of several pro-inflammatory mediatorsincluding TNF-α and finally protect tissues from TNF-α inducedapoptosis. TABLE 1 LCF Nourin Stimulates the Release of High Levels ofChemokines and Cytokines by Human Peripheral Monocytes Nourin ControlMedia Interleukin-8 12,000 ng/ml 2,000 ng/ml Interleukin-1β 400 pg/ml 10pg/ml Tumor Necrosis Factor-α 400 pg/ml 100 pg/ml

As will be apparent to persons skilled in the art, variousmodifications, adaptations and variations of the foregoing specificdisclosure can be made without departing from the teachings of thepresent invention as defined in the appended claims.

REFERENCES

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1. A method of treating mammalian tissue subject to injury, pain,ischemia, or infections agents, said method comprising administering tothe mammal at least one tissue-protective agent effective for impartingto the tissue an anti-inflammatory response, an anti-apoptotic response,or both anti-inflammatory and anti-apoptotic responses.
 2. The method ofclaim 1, wherein the agent comprises spinorphin, tynorphin, leuhistin,nimbidin, or a combination of more of these.
 3. The method of claim 1,wherein the agent comprises the peptide t-Boc-Phe-D-Leu-Phe-D-Leu-Phe(SEQ ID NO: 2) or t-Boc-Methionyl-Leucyl-Phenylalanine (SEQ ID NO: 5).4. The method of claim 1, wherein the agent comprisesCarbobenzoxy-Phe-Met (SEQ ID NO: 10), the Substance P antagonistR(dextro-)PKP (dextro-)FQ(dextro-)WF(dextro-)WLL-NH₂ (SEQ ID NO: 6), orCyclosporine H.
 5. The method of claim 1, wherein the agent comprises aplurality of soluble formyl peptide receptors that can bind toformylated ligands, non-formylated ligands, or both.
 6. The method ofclaim 1, wherein the agent comprises an antibody against formyl peptidereceptors that can bind to formylated ligands, an antibody againstformyl peptide receptors that can bind to non-formylated ligands, or anantibody against a tissue-derived leukocyte chemotactic factor.
 7. Themethod of claim 1, wherein the agent comprises an antagonist of atissue-derived leukocyte chemotactic factor.
 8. The method of claim 1,further comprising administering to the mammal at least one additionalagent selected from the group consisting of anti-oxidants, enzymes,enzyme inhibitors, antibiotics, inhibitors of the bacterial formylpeptides chemoattractants, and endogenous inhibitors of tissue-derivedleukocyte chemotactic factors.
 9. The method of claim 8, wherein theenzyme inhibitors comprise L-histidine, trasylol, or both L-histidineand trasylol.
 10. The method of claim 8, wherein the enzymes comprise atleast one deformylase.
 11. The method of claim 8, wherein theanti-oxidants comprise ascorbic acid.
 12. The method of claim 1, whereinthe agent comprises a creatine analogue.
 13. The method of claim 12,wherein the creatine analogue is cyclocreatine, a salt thereof, orcyclocreatine phosphate.
 14. The method of claim 1, wherein the agentcomprises nimbidin or a metabolite of mitochondria.
 15. The method ofclaim 14, wherein the metabolite is acetyl L-carnitine, coenzyme Q10,glutathione, or a-lipoic acid.
 16. The method of claim 1, furthercomprising administering to the mammal an additional agent that iscapable of generating nitric oxide in vivo.
 17. The method of claim 1,wherein the anti-inflammatory response arises from one or more actionsof the agent, said actions selected from the group consisting ofdelaying the depletion of adenosine triphosphate in the tissue,conserving the total adenylate pool in the tissue, buffering a decreasein the ratio of adenosine triphosphate to free adenosine diphosphate inthe tissue, delaying exhaustion of high-energy phosphates in the tissue,maintaining cell-membrane integrity in the tissue, inhibiting caspaseenzyme activity in the tissue, reducing intracellular edema in thetissue, and reducing lactic acidosis in the tissue.
 18. The method ofclaim 1, wherein the agent crosses the blood-brain barrier of themammal, accumulates in nerve tissue of the mammal, enhances energyproduction in the tissue, reduces lactic acidosis in the tissue, and/orreduces the level of malondialdehyde in the tissue.
 19. The method ofclaim 1, wherein the agent is administered prophylactically ortherapeutically during injury or post-injury.
 20. The method of claim 1,wherein the agent is administered by injection, orally, topically, or byinhalation.
 21. The method of claim 1, wherein the mammalian tissue ishuman tissue.
 22. The method of claim 1, wherein the injury is relatedto ischemia or infection.
 23. A composition for treating animal tissuesubject to injury, the composition comprising: (i) an anti-inflammatoryagent; and (ii) an anti-apoptotic agent, wherein the anti-inflammatoryagent is capable of inhibiting a tissue-derived leukocyte chemotacticfactor.
 24. The composition of claim 23, wherein the anti-inflammatoryagent comprises an antagonist of a tissue-derived leukocyte chemotacticfactor.
 25. The composition of claim 24, further comprising at least oneagent selected from the group consisting of anti-oxidants, enzymes,enzyme inhibitors, antibiotics, inhibitors of the bacterial formylpeptides chemoattractants, and endogenous inhibitors of tissue-derivedleukocyte chemotactic factors.
 26. The composition of claim 25, whereinthe enzyme inhibitors comprise L-histidine, trasylol, or bothL-histidine and trasylol.
 27. The composition of claim 25 wherein theenzymes comprise at least one deformylase.
 28. The composition of claim25, wherein the anti-oxidants comprise ascorbic acid.
 29. Thecomposition of claim 23, wherein the anti-inflammatory agent comprisesthe antagonist spinorphin, leuhistin, tynorphin, nimbidin, or acombination of two or all three of these; the synthetic compoundt-Boc-Phe-Leu-Phe-Leu-Phe (SEQ ID NO: 2); the immunosuppressivecyclosporine H; Carbobenzoxy-Phe-Met (SEQ ID NO: 10); a plurality ofsoluble formyl peptide receptors that can bind to formylated ligands; aplurality of soluble formyl peptide receptors that can bind tonon-formylated ligands; an antibody against formyl peptide receptorsthat can bind to formylated ligands; an antibody against formyl peptidereceptors that can bind to non-formylated ligands; an antibody against atissue-derived leukocyte chemotactic factor; Substance P antagonistR(dextro-)PKP(dextro-)FQ(dextro-)WF(dextro-)WLL-NH₂ (SEQ ID NO: 6); orcombinations of two or more of these.
 30. The composition of claim 23,wherein the anti-apoptotic agent comprises a creatine analogue.
 31. Thecomposition of claim 30, wherein the creatine analogue is cyclocreatineor a salt thereof, or cyclocreatine phosphate.
 32. The composition ofclaim 23, wherein the anti-apoptotic agent comprises a metabolite ofmitochondria.
 33. The composition of claim 32, wherein the metabolite isacetyl L-carnitine, coenzyme Q10, glutathione, or α-lipoic acid.
 34. Amethod of using a composition to protect or treat a first tissue of amammal, said first tissue suspected of being injured or of beingsusceptible to injury, comprising providing an effective amount of thecomposition to said first tissue, wherein the composition comprises (i)an anti-inflammatory agent and (ii) an anti-apoptotic agent, and whereinthe anti-inflammatory agent is capable of inhibiting a leukocytechemotactic factor derived from a second tissue of the mammal.
 35. Themethod of claim 34, wherein said first tissue and said second tissue arethe same.
 36. The method of claim 34, wherein the anti-inflammatoryagent comprises an antagonist of a tissue-derived leukocyte chemotacticfactor.
 37. The method of claim 36, wherein the composition furthercomprises at least one of the following agents: anti-oxidants, enzymes,enzyme inhibitors, antibiotics, inhibitors of the bacterial formylpeptides chemoattractants, and endogenous inhibitors of tissue-derivedleukocyte chemotactic factors.
 38. The method of claim 37, wherein theenzyme inhibitors comprise L-histidine, trasylol, or both L-histidineand trasylol.
 39. The method of claim 37, wherein the enzymes compriseat least one deformylase.
 40. The method of claim 37, wherein theanti-oxidants comprise ascorbic acid.
 41. The method of claim 36,wherein the antagonist comprises spinorphin, tynorphin, or both.
 42. Themethod of claim 41, wherein the antagonist further comprises leuhistin.43. The method of claim 34, wherein the anti-inflammatory agentcomprises nimbidin, the synthetic compound t-Boc-Phe-Leu-Phe-Leu-Phe(SEQ ID NO: 2), the immunosuppressive cyclosporine H,Carbobenzoxy-Phe-Met (SEQ ID NO: 10), a plurality of soluble formylpeptide receptors that can bind to formylated ligands, a plurality ofsoluble formyl peptide receptors that can bind to non-formylatedligands, an antibody against formyl peptide receptors that can bind toformylated ligands, an antibody against formyl peptide receptors thatcan bind to non-formylated ligands, an antibody against a tissue-derivedleukocyte chemotactic factor, Substance P antagonistR(dextro-)PKP(dextro-)FQ(dextro-)WF(dextro-)WLL-NH₂ (SEQ ID NO: 6), or acombination of two or more of these.
 44. The method of claim 34, whereinthe anti-apoptotic agent comprises a creatine analogue or a metaboliteof mitochondria.
 45. The method of claim 44, wherein the creatineanalogue is cyclocreatine or a salt thereof or cyclocreatine phosphate.46. The method of claim 44, wherein the metabolite is selected from thegroup consisting of acetyl L-carnitine, coenzyme Q10, glutathione, andα-lipoic acid.
 47. The method of claim 34, further comprisingadministering to the mammal an additional agent that is capable ofgenerating nitric oxide in vivo.
 48. A method of treating injuredmammalian tissue in a patient, said method comprising: (a) taking asample of patient mammalian tissue suspected of being damaged; (b)detecting the release of at least one protein from the mammalian tissue,to indicate that the mammalian tissue is in an injured state in thepatient; and (c) if the tissue is injured, administering to the mammalan effective amount of a composition comprising (i) an anti-inflammatoryagent and (ii) an anti-apoptotic agent, wherein the anti-inflammatoryagent is capable of inhibiting a tissue-derived leukocyte chemotacticfactor.
 49. A method of treating mammalian tissue subject to injury,said method comprising the step of administering to the mammal acreatine analogue in an amount between 0.03 g and 0.08 g creatineanalogue per kg of mammal body weight.
 50. The method of claim 49,wherein the creatine analogue reduces intracellular cAMP production inthe tissue.
 51. The method of claim 49, wherein the treatment reduces oreliminates apoptosis in the injured tissue.
 52. The method of claim 49,wherein the creatine analogue is cyclocreatine or a salt thereof orcyclocreatine phosphate.
 53. The method of claim 49, further comprisingadministering to the mammal an additional agent that is capable ofgenerating nitric oxide in vivo.